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标题: Inhibition of bicarbonate reabsorption in the rat proximal tubule by activation [打印本页]

作者: 轻羽 时间: 2009-4-22 08:09 标题: Inhibition of bicarbonate reabsorption in the rat proximal tubule by activation

作者：Matthew A. Bailey作者单位：Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520



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   The present study used a stationary microperfusion technique to investigate in vivo the effect of P2Y 1 receptor activation on bicarbonate reabsorption in the rat proximal tubule. Proximal tubules were perfused with a bicarbonate Ringer solution before flow was stopped by means of an oil block. The recovery of lumen pH from the initial value (pH 8.0) to stationary values (pH 6.7) was recorded by a H -sensitive microelectrode inserted downstream of the perfusion pipette and oil block. The stationary pH value and the t of pH recovery were used to calculate bicarbonate reabsorption ( J HCO3 ). Both EIPA and bafilomycin A1 caused significant reductions in proximal tubule J HCO3, consistent with the established contributions of Na/H exchange and H -ATPase to proximal tubule HCO 3 reabsorption. The nucleotides ADP and, to a lesser extent, ATP reduced J HCO3 but AMP and UTP were without effect. 2MeSADP, a highly selective agonist of the P2Y 1 receptor, reduced J HCO3 in a dose-dependent manner. MRS-2179, a P2Y 1 receptor-specific antagonist, abolished the effect of 2MeSADP, whereas theophylline, an antagonist of adenosine (P1) receptors, did not. The inhibitory action of 2MeSADP was blocked by inhibition of protein kinase C and reduced by inhibition of protein kinase A. The effects of EIPA and 2MeSADP were not additive. The data provide functional evidence for P2Y 1 receptors in the apical membrane of the rat proximal tubule: receptor activation impairs acidification in this nephron segment. ) Y; n8 L: y- a6 Z
      【关键词】 P receptor purinergic renal extracellular nucleotide
   PURINE AND PYRIMIDINE tri- and diphosphonucleotides are potent extracellular messengers that regulate cell function through the activation of specific cell-surface receptors called P2 receptors ( 7 ). This family divides on both structural and functional grounds into ionotropic P2X receptors (P2X 1-7 ) and metabotropic P2Y receptors (P2Y 1,2,4,6,11-14 ), which couple to the activation of phospholipase C ( 7 ), thereby increasing intracellular free calcium concentration ([Ca 2  ] i ).
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P2X and P2Y receptors have been identified in the mammalian kidney ( 2, 3, 20, 28, 31 ): multiple subtypes are often expressed in the same nephron segment, cell type, and even within the same membrane ( 19, 20 ). Given this complex distribution, renal cell lines are the paradigm through which the functional consequences of P2 receptor activation have largely been defined ( 20, 28 ). From this body of work, it is apparent that P2 receptor activation can modulate the activity of key renal transport proteins such as Na-K-ATPase ( 12 ), Na-K-2Cl cotransport ( 6, 12 ), and the amiloride-sensitive sodium channel ( 8, 22 ). In A6 cells, a cell line of distal tubule phenotype derived from Xenopus laevis, basolateral but not apical P2Y 1 receptor activation inhibits Na/H exchange ( 1 ). In the murine M1 cell line, which resembles the principal cell of the cortical collecting duct, the reverse applies: extracellular ATP reduces intracellular pH when applied to the apical but not basolateral membrane ( 16 ).- |/ }% Y! @1 M% T
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Given the potential for extracellular nucleotides to act as local mediators of transepithelial electrolyte flux, it is important to extend those observations made from renal cell models to the intact nephron. Thus far, such studies have focused on the distal nephron: basolateral P2Y 2 receptor activation inhibits AVP-stimulated osmotic water permeability in segments of the cortical and medullary collecting duct ( 20, 28 ), whereas activation of P2Y 2 receptors in the apical membrane inhibits small-conductance potassium channels ( 21 ) and amiloride-sensitive short-circuit current in the native mammalian collecting duct principal cell ( 18 ). In contrast, the physiological ramifications of purinergic signaling in the proximal tubule are largely unknown. Purinergic receptors are certainly expressed in this segment ( 2, 3, 31 ), although functional confirmation has been limited to the measurement of calcium transients following application of nucleotides to the basolateral membrane.6 e% t! ^2 N( S/ |* J, `
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Approximately 75% of filtered HCO 3 is reclaimed by the proximal tubule ( 15 ). The mechanism is indirect, involving H secretion across the apical membrane coupled with basolateral reabsorption of HCO 3 ( 15 ). The major fraction of apical H secretion is mediated by Na/H exchange, although H -ATPase also contributes ( 15 ). Because such transporters can be regulated by purinergic receptor activation ( 1, 29 ), the present study investigated the effect of luminally applied extracellular nucleotides on proximal tubule HCO 3 reabsorption in vivo.2 v" |( N, a* |- [" s
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METHODS

Male Sprague-Dawley rats ( n = 44; weight range 180-320 g) were anesthetized with an intraperitoneal injection of thiobutabarbital sodium (Inactin 100 mg/kg; Sigma) and prepared surgically for in vivo micropuncture experiments: the left jugular vein and carotid artery were cannulated for infusion of isotonic saline (3 ml/h) and blood sampling, respectively. A tracheotomy was performed and the bladder was catheterized for urine collection. The left kidney was exposed by flank incision and immobilised in a Perspex cup clamped to the operating table. During the experiments, body temperature was maintained at 37°C.

Stationary microperfusion of the proximal tubule. The technique employed in these experiments has been described in detail previously ( 23, 32 ). A proximal tubule was impaled by a perfusion micropipette and injection of FD&C green-colored saline was used to identify downstream loops of the proximal tubule. A H -selective microelectrode was lowered onto the kidney surface for zero-voltage measurement before being inserted into the proximal tubule lumen downstream of the perfusion pipette. A second micropipette containing Sudan black-colored heavy mineral oil was inserted into a tubular segment between the perfusion pipette and microelectrode. For stationary measurements of proximal tubular H secretion, the inserted perfusion pipette was used to inject a control solution into the tubule lumen until the electrode recorded a large and stable voltage deflection. At this point, perfusion was stopped and a column of oil was injected to prevent downstream flow of either native tubule fluid or perfusate. Voltage measurements were recorded until a stable reading was once again obtained. After control measurements, the first pipette was replaced by a second perfusion pipette containing the experimental perfusion solution (i.e., the control solution to which a chemical agent had been added). Recordings were again made. In some instances, the control pipette was reinserted to provide time control data. In a typical experiment, each nephron was perfused first with the control solution (2-3 recordings) and then with at least one experimental solution (4-6 recordings), allowing for paired assessment of H secretion. In each animal, two to nine nephrons were perfused in the manner described.# U- O. B; B1 R( I% S+ m

Solutions and chemicals. The control perfusate contained NaCl (100 mmol/l) and NaHCO 3 (25 mmol/l). Raffinose pentahydrate (Riedel-de Haën, Hannover, Germany) was added to the perfusate to establish isosmolality with plasma and thereby minimize fluid reabsorption. Under these equilibrium conditions, increases in lumen H activity relate directly to tubular H secretion ( 23 ). For experimental solutions, the following compounds were used (all from Sigma): EIPA (0.1% DMSO vehicle); bafilomycin A1 (0.1% DMSO vehicle); ATP; ADP; AMP; UTP; 2-methylthioadenosine 5'-diphosphate (2MeSADP); theophylline (0.1% DMSO vehicle); N

6 )-methly-2'-deoxyadenosine-3',5'-bisphosphate (MRS-2179); N -(2-[ p -bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H-89); and 1-{6-[([17 ]-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl}-1H-pyrrole-2,5-dione (U-73122; 0.05% DMSO vehicle).

Ion-selective microelectrodes. Double-barreled microelectrodes were pulled from asymmetric borosilicate glass to a tip diameter of 1 µm. The smaller reference barrel was backfilled with a solution containing 0.24 M NaCl and 0.76 M Na acetate, colored by FD&C green. The cation/anion mobility of this solution was the same because the mobility in solution of chloride is larger than that of sodium, whereas that of acetate is smaller. The rat was electrically grounded through the tail, and measurements of transepithelial potential difference were made through the reference barrel., v/ ~6 X2 a7 P. }% e' P

The larger barrel was exposed for 30 min at room temperature to hexamethyldisilazane (Fluka, Buchs, Switzerland) before the tip was filled with H ionophore ( cocktail B; Fluka). The electrode was calibrated before and after impalement by superfusion onto the kidney surface of standards kept at 37°C. Standards of pH 6.0, 7.0, and 8.0 were used to measure the mean voltage difference per 10-fold change in H activity. This was 52.7 ± 0.5 mV ( n = 159 electrodes). The voltages from each barrel were measured by a high-impedance electrometer (model 223, World Precision Instruments, Sarasota, FL). An analog-to-digital converter (model 104, iWorx, Dover, NH), sampling at a rate of 5 Hz, allowed voltages to be recorded onto a PC using LabScribe software (iWorx).% P) q, y% X0 C+ t. j
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Analysis. Free-flow transepithelial potential difference recorded by the reference electrode ranged from -3 to  4 mV; the contribution of this voltage to that recorded by the H -selective channel was nugatory. The voltage difference ( Fig. 1 A ) between the reference and ion-selective barrels, representing lumen H activity, was converted initially to pH ( Fig. 1 B ). For each perfusion, pH fell from the initial value of 8 to a stationary value (pH stat ) of 6.8. A one-phase exponential was fitted to this fall, allowing the half-time ( t ) of proximal acidification to be generated. Bicarbonate reabsorption ( J HCO3 ) was then calculated using the following equation ( 32 )
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where t is the acidification half-time, initial HCO 3 is 25 mmol/l, stationary HCO 3 is calculated from stationary pH and arterial P CO 2, and r is the lumen radius. Arterial P CO 2 was 43.5 ± 0.9 mmHg. It should be noted that acidification measured under the present conditions consists of two components: acidification due to equilibration of the lumen perfusate with cortical CO 2 and acidification due to proton secretion by the proximal tubule ( 13, 23 ). Both of these components will, in the first instance, occur concomitantly. CO 2 equilibration is, however, both rapid and transient, being effectively complete within the first seconds of measurement. Thus the bicarbonate reabsorption reported here includes a small equilibration flux, assumed to be constant within the paired perfusions.* |9 B8 w/ w: V( Z8 r4 ^8 T: O4 R

Fig. 1. Example of the data recording and transformation during perfusion of the same proximal tubule with control solution (solid line) and a solution containing 2MeSADP (100 µmol/l; dashed line). After perfusion (P) with the solution at pH 8 and block (B), H activity (mV) increases. Conversion to pH ( B ) allows the t of acidification from initial pH to stationary pH (pH stat ) to be measured: these values are used to calculate J HCO3.

Statistics. Data are presented as means ± SE obtained from individual recordings under a given condition. Statistical comparisons were made using one-way ANOVA; the probability of 0.05 was taken as the limit of statistical significance. Specified point-to-point comparisons were made using Bonferroni's test.

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Effect of EIPA and bafilomycin A1. The methodology employed here yields values of bicarbonate reabsorption calculated from the rate and extent of lumen fluid acidification. Because bicarbonate reabsorption in the rat proximal tubule is largely mediated by Na/H exchange and vacuolar H -ATPase ( 15 ), the present technique was validated using luminal application of inhibitors of these transporters, EIPA (200 µmol/l) and/or bafilomycin A1 (1 µmol/l), respectively. As shown in Fig. 2, each agent significantly impaired J HCO3. For EIPA, the inhibitory effect reflects significant reductions in the both the rate of acidification ( t ) and the maximum pH gradient achieved. For bafilomycin A1, although pH stat was significantly higher than in controls, the increase in t did not reach statistical significance. The inhibitory effect on J HCO3 of perfusion with the two inhibitors together was significantly greater than perfusion with either EIPA or bafilomycin A1 alone. The inhibitory effects on J HCO3 recorded during blockade of Na/H exchange and/or H ATPase were similar in magnitude to data obtained in the rat and mouse using direct quantitative measurement of bicarbonate flux ( 34, 35 ). The time control data were not different from the control data.
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Fig. 2. A : example recording showing acidification during paired perfusion with control (solid line) and EIPA (dashed line). B : J HCO3 during perfusion with control ( n = 143) and either time control (0.1% DMSO, n = 44), EIPA (200 µmol/l, n = 52), bafilomycin A1 (BAF; 1 µmol/l, n = 44), or EIPA BAF ( n = 41). C : t of pH recovery. D : stationary pH. Data are means ± SE of individual recordings taken from 46 tubules in 17 rats. ** P ) d" n- i) j$ _9 s) D

Effect of adenine and uridine nucleotides. A sample recording obtained during perfusion of the lumen with either ATP or ADP (both at 100 µmol/l) is shown in Fig. 3 A. In Fig. 3 B, the actions of AMP, ADP, and ATP on J HCO3 are shown: AMP was without effect, whereas both ADP ( P 4 C7 g- R" o2 j

Fig. 3. A : example recording showing acidification during paired perfusion with control (solid line) and either ATP (dashed line) or ADP (solid grey line). B : J HCO3 during perfusion with control ( n = 67) and either AMP (100 µmol/l; n = 30), ADP (100 µmol/l; n = 40), or ATP (100 µmol/l; n = 20). Data are means ± SE of all individual recordings taken from 22 tubules in 4 rats; each tubule was perfused with control solution and then again with 1 experimental solution. * P
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Activation of P2Y 1 receptors. A sample recording showing the effect of perfusion with 100 µM 2MeSADP, a potent and selective P2Y 1 receptor agonist ( 27 ), is shown in Fig. 4 A. This agent reduced J HCO3 by 50%, as shown in Fig. 4 B. Most of this effect can be attributed to a reduction in t ( Fig. 4 C ), i.e., in the rate of acidification, although pH stat was significantly higher during perfusion with 2MeSADP than under control conditions ( P ' X7 V/ z- s! [: Y& R

Fig. 4. A : example recording showing acidification during paired perfusion with control (solid line) and 2MeSADP (100 µmol/l; dashed line). B : J HCO3 during perfusion with control ( n = 113) and either time control (saline, n = 52) or 2MeSADP ( n = 88). C : t of pH recovery. D : stationary pH. Data are means ± SE of individual recordings taken from 31 tubules in 11 rats. ** P $ T7 R& }. [0 [% Q0 e: v6 }9 \

Fig. 5. J HCO3 during perfusion of the same tubule with control and 4 successive applications of 2MeSADP. Data are means ± SE of individual recordings. ** P 0 A9 @) \6 E$ p% {

These data suggest that P2Y 1 receptors exert a controlling influence on proximal tubule bicarbonate reabsorption. Nevertheless, alternative hypotheses must be considered. First, commercial preparations of 2MeSADP are contaminated with small amounts of 2MeSAMP and thus the actions attributable to P2Y 1 receptor agonism could, in fact, reflect actions on the P1 receptor family. To assess this possibility, theophylline, a potent nonspecific antagonist of P1 receptors, was added to the perfusate. Theophylline (1 mmol/l) alone had no effect on J HCO3. Nor did it attenuate the inhibitory effect of 100 µM 2MeSADP ( Fig. 6 ), even at this high concentration.$ L! J2 e6 _& J6 F( D( E) z

Fig. 6. J HCO3 during P1 receptor blockade. Tubules were perfused with control ( n = 71) and either theophylline (Theo; 1 mmol/l; n = 20), 2MeSADP (100 µmol/l; n = 49), or both agents together ( n = 48). Data are means ± SE of individual recordings taken from 21 tubules in 5 rats. ** P

Second, 2MeSADP can activate (albeit with very low potency) P2Y 6 receptors ( 7 ), mRNA for which is found in rat proximal tubule ( 3 ). To assess this possibility, a selective, competitive inhibitor of the P2Y 1 receptor, MRS-2179 ( 5 ), was added to the perfusate ( Fig. 7 ): at a concentration of 1 mmol/l, MRS-2179 increased J HCO3 significantly, suggesting basal activation of lumen P2Y 1 receptors by endogenous adenine nucleotides. In addition, MRS-2179 completely blocked the inhibitory effect of 2MeSADP. Identical results were obtained with a lower concentration (200 µmol/l) of MRS-2179 (data not shown). This observation confirms that the inhibition of J HCO3 by 2MeSADP is mediated by P2Y 1 receptors.0 A5 E2 j9 Q+ i1 z9 A
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Fig. 7. J HCO3 during P2Y 1 receptor blockade. A : example recording showing acidification during paired perfusion with control (solid line) and MRS-2179 (1 mmol/l; dashed line). B : J HCO3 during perfusion with control ( n = 101) and either MRS-2179 ( n = 37), 2MeSADP (100 µmol/l; n = 47), or both agents together ( n = 45). C : t of pH recovery. D : stationary pH. Data are means ± SE of individual recordings taken from 32 tubules in 8 rats. ** P ' Z2 u& {9 d" G, T
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Concentration-response curve. Figure 8 shows the effect of perfusion with 2MeSADP at concentrations between 100 nmol/l and 1 mmol/l. Because only one concentration of agonist was used per tubule, the data are shown as percentage inhibition of the paired control value. 2MeSADP caused a significant reduction in bicarbonate reabsorption at luminal concentrations of 1 µmol/l or greater and maximum inhibition was effectively achieved at a lumen concentration of 10 µmol/l 2MeSADP.
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Fig. 8. J HCO3 during paired perfusion with control and 2MeSADP at either 0.1 ( n = 28), 1 ( n = 49), 10 ( n = 61), 100 ( n = 88), or 1,000 ( n = 35) µmol/l. Data are means ± SE of individual recordings taken from 53 tubules in 14 rats and are transformed to show the percentage inhibition of paired control values. ** P
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Intracellular signaling pathways. The rat P2Y 1 receptor couples to G q protein and, via phospholipase C, leads to an increase in [Ca 2  ] i and activation of protein kinase C ( 26 ). In the present study, inhibition of the phospholipase C pathway using U-73122 (10 µmol/l), a lipid-soluble agent that inhibits PPI hydrolysis by phospholipase C in a dose-dependent manner ( 36 ), inhibited fully the action of 2MeSADP (100 µmol/l) on proximal bicarbonate reabsorption ( Fig. 9 A ). Perfused alone, U-73122 had no effect, suggesting low basal activity phospholipase C under the present conditions.& q5 \% n& S9 S. U$ B$ Z
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Fig. 9. J HCO3 during perfusion with ( A ) control ( n = 50), U-73122 (10 µmol/l; n = 45), 2MeSADP (100 µmol/l; n = 36), or both agents together ( n = 52) and ( B ) control ( n = 99), H89 (10 µmol/l; n = 25), 2MeSADP (100 µmol/l; n = 40), or both agents together ( n = 65). Data are means ± SE of individual recordings taken from 37 tubules in 9 rats. ** P " X: K7 x; k) L
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The possible role of protein kinase A was assessed in the present study by measuring the impact of H-89, a lipid-soluble inhibitor of the kinase ( 11 ), on the actions of 2MeSADP. As seen in Fig. 9 B, H-89 (10 µmol/l) alone tended to increase bicarbonate reabsorption in the proximal tubule, although this did not reach statistical significance. There was a significant reduction in J HCO3 during combined perfusion with 2MeSADP and H-89 compared with perfusion with H-89 alone. Nevertheless, under these conditions, J HCO3 was significantly greater than observed with 2MeSADP alone.9 |6 H" R; ?2 q0 T2 V% F

Mechanism of inhibition. To investigate the mechanism by which P2Y 1 receptor activation evoked reduced bicarbonate reabsorption, the proximal tubule was perfused in one series of experiments with three separate solutions: control, EIPA (200 µmol/l), and EIPA (200 µmol/l) plus 2MeSADP (100 µmol/l). The effect of a maximum inhibitory concentration of 2MeSADP was not additive to that of a maximum inhibitory concentration of EIPA ( Fig. 10 ). This indicates that P2Y 1 receptor activation impairs bicarbonate reabsorption in the proximal tubule via inhibition of Na/H exchange.

Fig. 10. J HCO3 during paired perfusion of the same proximal tubule with control ( n = 49), EIPA (200 µmol/l; n = 52), and EIPA 100 µmol 2MeSADP ( n = 63). Data are means ± SE of individual recordings taken from 28 tubules in 7 rats. ** P $ B* L: p4 ~- l( m& U0 O2 z
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DISCUSSION
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The rat proximal tubule expresses mRNA of four members of the P2Y receptor family ( 2, 3 ), three of which (P2Y 2, P2Y 4, and P2Y 6 ) are pyrimidine selective. The fourth, P2Y 1, is activated by purine nucleotides, particularly ADP ( 7 ). The confirmation of functional receptor expression through measurement of nucleotide-stimulated increases in [Ca 2  ] i has thus far been limited to the basolateral membrane where P2Y 1 is the predominant receptor subtype ( 2 ). Recent immunohistological data indicate that P2Y 1 receptors are also expressed in the apical membrane of the rat proximal tubule ( 31 ): the present investigation demonstrates a functional role for apical P2Y 1 receptors and suggests that luminal nucleotides are modulators of transepithelial bicarbonate transport.

The proximal tubule reclaims 75% of filtered bicarbonate through Na/H exchanger- and H -ATPase-mediated proton secretion ( 15 ): both transporters are regulated by extracellular nucleotides in renal and nonrenal cell lines ( 1, 12, 16, 24, 29 ). The present study extended those observations made in isolated cells to a more complex biological system. The observed inhibition of bicarbonate reabsorption by the endogenous nucleotides ADP and, to a lesser extent, ATP, strongly implicates P2Y 1 receptors in the regulation of proximal tubule transport processes. This supposition was confirmed by two observations: first, the greatest degree of inhibition was observed using 2MeSADP, the most potent agonist of P2Y 1 receptors ( 27 ). Second, 2MeSADP was no longer able to inhibit J HCO3 in the presence of the P2Y 1 receptor-specific antagonist, MRS-2179. Immunohistological data suggest that the only other P2 receptor expressed in the apical membrane of the proximal tubule is P2X 5 ( 31 ), at which 2MeSADP is not an agonist ( 7 ). P2X 5 is activated by UTP ( 7 ), but this agent was without effect on J HCO3.3 L/ |. e% k- `, C( w. V; ]
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At maximal activation, P2Y 1 receptors reduce proximal tubule bicarbonate reabsorption by 50%. This occurs at a lumen nucleotide 100 µmol/l, so it is doubtful whether such extensive inhibition is physiological. Moreover, because the proximal tubule is a leaky epithelium, it is possible that high concentrations of nucleotide gain access to sites other than the apical membrane.! s9 q; h) q  W: t
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Of greater physiological importance is the submaximal inhibition of proximal tubule bicarbonate reabsorption occurring between 1 and 10 µM 2MeSADP; at these concentrations, it is unlikely that significant leakage to the basolateral membrane could occur. Moreover, such concentrations of endogenous receptor agonist might be present in the tubule fluid. It is known, for example, that ATP is released across the apical membrane of isolated proximal tubule cells in quantities sufficient to achieve local concentrations in the low-micromolar range ( 28 ). Preliminary free-flow micropuncture studies indicate that the concentration of ATP in fluid samples harvested from the proximal tubule is 250 nmol/l ( 28a ), which, given the susceptibility of ATP to enzymatic degradation by proximal tubule ectonucleotidases ( 17 ), might underestimate significantly the actual concentration of nucleotide present at the apical membrane. Finally, it must be noted that the concentration of ADP in proximal tubule fluid has not yet been established: ATP, itself a P2Y 1 receptor agonist, provides a source for the more potent agonist ADP, thereby prolonging the regulation of proximal transport through P2Y 1 receptor activation.- F& @4 n) A3 @% T! o0 j

In heterologous expression systems, the rat P2Y 1 receptor couples exclusively to activation of phospholipase C ( 26 ); inhibition of this pathway blocked the actions of 2MeSADP in the present study. Activation of protein kinase C inhibits NHE3 in transfected cells ( 12 ) and reduces bicarbonate ( 25 ) and volume ( 4 ) reabsorption in the microperfused proximal tubule. This latter finding is not universal, however, and may relate to the conditions of exposure ( 33 ) and the basal levels of protein kinase C.
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The current study also advocates a role for protein kinase A as H-89 attenuated the effect of P2Y 1 receptor activation. Data obtained from A6 cells reveal direct inhibition of NHE3 by a cAMP/protein kinase A-dependent pathway ( 1 ). However, direct coupling of P2Y 1 receptors to G q or G s proteins is not evidenced ( 26 ), and thus the activation of protein kinase A by P2Y 1 receptor activation may well be indirect, resulting from regulation of adenylate cyclase or phosphodiesterase by [Ca 2  ] i ( 9 ).
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The nonadditive effects of 2MeSADP and EIPA suggest purinergic modulation of proximal tubule Na/H exchange and point, therefore, to inhibition of NHE3. Mechanistically, this could relate to a direct action on NHE3 by protein kinase A and/or protein kinase C, as stated above. This claim is buttressed by the fact that mutation of Ser 552 and Ser 605 in the rat NHE3 protein, which prevents phosphorylation of the transporter by protein kinase A ( 37 ), eliminates the inhibitory action of P2Y 1 receptor activation ( 1 ). Indirect effects on Na/H exchange may involve modulation of basolateral transporters such as the Na-K-ATPase or Na/HCO 3 cotransporter ( 30 ) by P2Y 1 receptor activation and purinergic effects on carbonic anhydrase have also been reported ( 14 ).& Y5 W" C' T* ]: o) H8 D3 w2 [. V

In summary, luminal P2Y 1 receptor activation inhibits bicarbonate reabsorption in the rat proximal tubule through a phospholipase C- and protein kinase A-dependent mechanism. The physiological ramifications of this inhibition are unresolved, but local control of NaHCO 3 transport is implicated. One possibility is that coordinated activation of purinergic receptors in both apical and basolateral domains contributes to glomerulotubular balance processes. Increased filtration at the glomerulus leads to a concatenation of events that would stimulate NaHCO 3 reabsorption to match filtered load: increased peritubular fluid viscosity promotes transepithelial bicarbonate flux, dependent on activation of basolateral P2 receptors ( 10 ), and increased tubular flow has been suggested to exert a diluting effect on luminal nucleotide ( 19 ), thereby alleviating inhibition of NaHCO 3. Such events remain hypothetical. Nevertheless, the present study adds to the growing body of evidence advocating purinergic regulation of renal function.1 t; I; k- _! |9 M( `
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GRANTS
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The author was supported by a Wellcome Trust International Fellowship.
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ACKNOWLEDGMENTS

I am grateful to G. Giebisch for advice and encouragement. I also thank S. Hebert, G. Malnic, and G. MacGregor for discussion of the data and D. Shirley, G. Burnstock, and R. Unwin for criticism of the manuscript0 z5 s+ l8 u) a' P
      【参考文献】0 k3 x9 y9 h7 C9 `9 @# [' I
Bagorda A, Guerra L, Di Sole F, Helmle-Kolb C, Favia M, Jacobson KA, Casavola V, and Reshkin SJ. Extracellular adenine nucleotides regulate Na /H exchanger NHE3 activity in A6-NHE3 transfectants by a cAMP/PKA-dependent mechanism. J Membr Biol 188: 249-259, 2002.
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Bailey MA, Imbert-Teboul M, Turner C, Marsy S, Srai K, Burnstock G, and Unwin RJ. Axial distribution and characterization of basolateral P2Y receptors along the rat renal tubule. Kidney Int 58: 1893-1901, 2000.

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Bailey MA, Imbert-Teboul M, Turner C, Srai SK, Burnstock G, and Unwin RJ. Evidence for basolateral P2Y 6 receptors along the rat proximal tubule: functional and molecular characterization. J Am Soc Nephrol 12: 1640-1647, 2001.

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Baum M and Hays SR. Phorbol myristate acetate and dioctanoylglycerol inhibit transport in rabbit proximal convoluted tubule. Am J Physiol Renal Fluid Electrolyte Physiol 254: F9-F14, 1988.

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Boyer JL, Romero-Avila T, Schachter JB, and Harden TK. Identification of competitive antagonists of the P2Y 1 receptor. Mol Pharmacol 50: 1323-1329, 1996.& p( `! B' N' l% _, e! q
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- R' m1 J  \' X3 z6 E0 \# ?

Brindikova TA, Bourcier N, Torres B, Pchezhetski D, Gekle M, Maximov GV, Montminy V, Insel PA, Orlov SN, and Isenring P. Purinergic-induced signaling in C11-MDCK cells inhibits the secretory Na-K-Cl cotransporter. Am J Physiol Cell Physiol 285: C1445-C1453, 2003.

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& g1 e( d0 [2 z+ j) d8 g
Burnstock G. ATP and its metabolites as potent extracellular agents. In: Current Topics in Membranes 54. Extracellular Nucleotides and Nucleosides: Release, Receptors, and Physiological & Pathophysiological Effects, edited by Schwiebert EM. New York: Academic, 2003, p. 1-27.

Cuffe JE, Bielfeld-Ackermann A, Thomas J, Leipziger J, and Korbmacher C. ATP stimulates Cl - secretion and reduces amiloride-sensitive Na absorption in M-1 mouse cortical collecting duct cells. J Physiol 524: 77-90, 2000.
" b; Z. G. s" O' ]: i* ?
9 `2 |  p% y( c4 l6 E8 d6 i+ k

Defer N, Best-Belpomme M, and Hanoune J. Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase. Am J Physiol Renal Physiol 279: F400-F416, 2000.8 ]  [. x) {7 F0 D

, i, ^) X  e* y7 S+ D

Diaz-Sylvester P, Mac Laughlin M, and Amorena C. Peritubular fluid viscosity modulates H flux in proximal tubules through NO release. Am J Physiol Renal Physiol 280: F239-F243, 2001.9 |4 p" g! l4 \, V

Fujihara M, Muroi M, Muroi Y, Ito N, and Suzuki T. Mechanism of lipopolysaccharide-triggered junB activation in a mouse macrophage-like cell line (J774). J Biol Chem 268: 14898-14905, 1993.
8 `. n- t' o! |) h( g7 [/ T
& P, N# O) J4 x1 I2 I9 z% X/ |
8 V; K1 X6 ~- a9 P! N/ z8 M) ~
Gagnon F, Hamet P, and Orlov SN. Na  ,K pump and Na -coupled ion carriers in isolated mammalian kidney epithelial cells: regulation by protein kinase C. Can J Physiol Pharmacol 77: 305-319, 1999.7 z5 m4 N8 z, E' R
9 L/ U3 _7 S" z2 j
. E( [, t' H/ c0 B4 }7 \- {
, C( g+ U+ X9 H; {) S
Giebisch G, Malnic G, De Mello GB, and De Mello Aires M. Kinetics of luminal acidification in cortical tubules of the rat kidney. J Physiol 267: 571-599, 1977.) ~* @; w' v% E) b6 T4 |5 g; [7 j9 O

; X( z) D' i3 v  I# I! @& s
! [4 [/ [8 Y3 Z# j  B
Glombitza S, Dragon S, Berghammer M, Pannermayr M, and Baumann R. Adenosine causes cAMP-dependent activation of chick embryo red cell carbonic anhydrase and 2,3-DPG synthesis. Am J Physiol Regul Integr Comp Physiol 271: R973-R981, 1996.
  B0 `; D9 O  @  m6 y$ ?. C
3 T, d6 q4 l6 X  Z% j. m. x

Hamm LL. Renal acidification mechanisms. In: The Kidney, edited by Brenner BM. Philadelphia, PA: Saunders, 2004, p. 497-534.# ~: S+ e2 p- t7 d1 U( D

" u# ~# Q' _) m3 B4 C6 ^% h( d
Hill C, Giesberts AN, and White SJ. Expression of isoforms of the Na /H exchanger in M-1 mouse cortical collecting duct cells. Am J Physiol Renal Physiol 282: F649-F654, 2002.8 Y9 S5 M+ s9 r

Le Hir M and Kaissling B. Distribution and regulation of renal ecto-5'-nucleotidase: implications for physiological functions of adenosine. Am J Physiol Renal Fluid Electrolyte Physiol 264: F377-F387, 1993.% l* Y9 u0 L0 e
  @" m5 w8 h& I( T( Y) K7 N
& @  H" {- d3 d$ k/ p
2 {& S% {( K* m2 A5 X
Lehrmann H, Thomas J, Kim SJ, Jacobi C, and Leipziger J. Luminal P2Y 2 receptor-mediated inhibition of Na absorption in isolated perfused mouse CCD. J Am Soc Nephrol 13: 10-18, 2002.# c/ {4 |7 A* l3 _6 `' ^8 Y

Leipziger J. Control of epithelial transport via luminal P2 receptors. Am J Physiol Renal Physiol 284: F419-F432, 2003.

Leipziger J, Bailey MA, and Unwin RJ. Purinergic receptors in the kidney. In: Current Topics in Membranes 54. Extracellular Nucleotides and Nucleosides. Release, Receptors, and Physiological and Pathophysiological Effects, edited by Schwiebert EM. New York: Academic, 2003, p. 369-394.

8 T/ n/ C5 g+ ?* c  Y- o

Lu M, MacGregor GG, Wang W, and Giebisch G. Extracellular ATP inhibits the small-conductance K channel on the apical membrane of the cortical collecting duct from mouse kidney. J Gen Physiol 116: 299-310, 2000.
0 B! U! l6 y7 z$ }6 i. u; n/ |  {

Ma HP, Li L, Zhou ZH, Eaton DC, and Warnock DG. ATP masks stretch activation of epithelial sodium channels in A6 distal nephron cells. Am J Physiol Renal Physiol 282: F501-F505, 2002.! v8 S9 C! G# N; ^- g9 Z# B, m, u
: u$ b# v; l* X" N: h) ~
9 t' |5 X0 H+ R0 e$ ?8 B
4 M: }% f+ W% C& L3 L5 h2 x
Malnic G and de Mello-Aires M. Kinetic study of bicarbonate reabsorption in proximal tubule of the rat. Am J Physiol 220: 1759-1767, 1971.2 r  W7 B- R( W

) D- U# f* p1 v* r% _

Okada Y, Taniguchi T, Akagi Y, and Muramatsu I. Two-phase response of acid extrusion triggered by purinoceptor in Chinese hamster ovary cells. Eur J Pharmacol 455: 19-25, 2002.
' R' C6 \" s' C3 l
( ^  y+ {  Y5 X% u
2 {9 Q( n8 p7 ~- i" K8 K; E; }4 \" P
Reboucas NA and Malnic G. Regulation of rat proximal tubule Na/H exchange by protein kinase C. Kidney Blood Press Res 19: 87-93, 1996.* j$ S* o+ |; O; V# O

$ g& P2 l: m% j1 ^

Schachter JB, Boyer JL, Li Q, Nicholas RA, and Harden TK. Fidelity in functional coupling of the rat P2Y 1 receptor to phospholipase C. Br J Pharmacol 122: 1021-1024, 1997." @" Z. p& W& d: Q2 F$ `# S& `

9 }* F" p: x# v

Schachter JB, Li Q, Boyer JL, Nicholas RA, and Harden TK. Second messenger cascade specificity and pharmacological selectivity of the human P2Y 1 -purinoceptor. Br J Pharmacol 118: 167-173, 1996.

9 |% P+ b) L2 j( j5 y+ l
; T$ F7 Z  b4 _8 |$ U
Schwiebert EM and Kishore BK. Extracellular nucleotide signaling along the renal epithelium. Am J Physiol Renal Physiol 280: F945-F963, 2001.

Shirley DG, Vekaria RM, and Unwin RJ. Proximal and distal tubular fluid ATP concentration in vivo (Abstract). J Am Soc Nephrol 13: A0072, 2003.
  @& E2 J9 ~: e" d7 Y- a
0 H7 Z8 @7 d4 g( J5 ]% E+ U* A; J
1 C5 v/ S* K2 r. @# l+ b
Susztak K, Kaldi K, Kapus A, and Ligeti E. Ligands of purinergic receptors stimulate electrogenic H -transport of neutrophils. FEBS Lett 375: 79-82, 1995.& T0 w( P" P) C9 g% ?1 G6 i+ x# ]: Y

6 C1 k5 N# G! R( Y/ t
8 g! |+ h( K, k, G1 J, b& D
Takeda M, Yoshitomi K, and Imai M. Regulation of Na -3HCO 3 - cotransport in rabbit proximal convoluted tubule via adenosine A1 receptor. Am J Physiol Renal Fluid Electrolyte Physiol 265: F511-F519, 1993.
( K' k7 d0 [0 m1 {' v
$ E  l+ {# ~0 A% J0 ~

Turner CM, Vonend O, Chan CM, Burnstock G, and Unwin RJ. The pattern of distribution of selected ATP-sensitive P2 receptor subtypes in rat kidney: an immunohistochemical study. Cells Tissues Organs 175: 105-117, 2003.* s. @7 q; }' S: X
5 o& R$ _" u9 i: R) F' G  I9 b1 o9 j0 D
! d5 a( p9 R5 ]# G9 y1 O1 Z; O

Ulate G, Fernandez R, and Malnic G. Effect of bafilomycin on proximal bicarbonate absorption in the rat. Braz J Med Biol Res 26: 773-777, 1993.

! x7 f; _# O( O2 F' S3 E3 [+ U
Wang T and Chan YL. Time- and dose-dependent effects of protein kinase C on proximal bicarbonate transport. J Membr Biol 117: 131-139, 1990.$ o" z" `+ X& I) o

2 T' K7 A. N) M9 `$ y+ {
/ l; r3 c) _( o3 @0 U' n$ D
Wang T, Hropot M, Aronson PS, and Giebisch G. Role of NHE isoforms in mediating bicarbonate reabsorption along the nephron. Am J Physiol Renal Physiol 281: F1117-F1122, 2001.' Y( p5 s0 @. X/ m
, ^  Q9 M. H' X$ C) m$ Q) X/ n
. A& @' k3 b' U
! H# T6 h( ~$ E* X, R& w
Wang T, Yang CL, Abbiati T, Schultheis PJ, Shull GE, Giebisch G, and Aronson PS. Mechanism of proximal tubule bicarbonate absorption in NHE3 null mice. Am J Physiol Renal Physiol 277: F298-F302, 1999.2 [& B+ ~* l2 K6 }- S& m
0 n1 h8 ^6 B+ a

Yule DI and Williams JA. U73122  inhibits Ca 2  oscillations in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells. J Biol Chem 267: 13830-13835, 1992.
, Z  u( r) b. z; Q
2 ^$ O8 l  `6 Z! ^' m
0 I2 ?+ Z! Z4 T
Zhao H, Wiederkehr MR, Fan L, Collazo RL, Crowder LA, and Moe OW. Acute inhibition of Na/H exchanger NHE-3 by cAMP. Role of protein kinase A and NHE-3 phosphoserines 552 and 605. J Biol Chem 274: 3978-3987, 1999.

作者: xuguofeng 时间: 2015-5-30 12:43

文笔流畅，修辞得体，深得魏晋诸朝遗风，更将唐风宋骨发扬得入木三分，能在有生之年看见楼主的这个帖子。实在是我三生之幸啊。

作者: MIYAGI 时间: 2015-6-18 12:38

病毒转染干细胞

作者: 罗马星空 时间: 2015-8-12 12:51

这样的贴子，不顶说不过去啊

作者: 大小年 时间: 2015-8-14 16:27

(*^__^*) 嘻嘻……

作者: sky蓝 时间: 2015-8-21 07:40

我十目一行也还是看不懂啊

作者: 我心飞翔 时间: 2015-10-22 12:49

今天没事来逛逛

作者: 舒思 时间: 2015-11-1 16:59

支持一下

作者: 舒思 时间: 2015-11-4 21:10

干细胞治疗糖尿病

作者: 石头111 时间: 2015-11-9 11:43

帮你顶，人还是厚道点好

作者: 兔兔 时间: 2015-11-22 19:54

细胞治疗行业

作者: 石头111 时间: 2015-12-10 22:17

支持你一下下。。

作者: 命运的宠儿 时间: 2015-12-30 11:39

支持~~

作者: txxxtyq 时间: 2016-1-22 18:41

@,@..是什么意思呀？

作者: tuanzi 时间: 2016-2-7 13:14

厉害！强~~~~没的说了！

作者: lalala 时间: 2016-2-13 16:34

这个贴好像之前没见过

作者: xiao2014 时间: 2016-3-4 15:43

顶你一下，好贴要顶！

作者: 知足常乐 时间: 2016-4-23 18:16

干细胞研究还要面向临床

作者: 小倔驴 时间: 2016-5-11 16:53

希望大家都有好运

作者: nauticus 时间: 2016-5-13 08:43

又看了一次

作者: 3344555 时间: 2016-5-14 16:42

神经干细胞

作者: dogcat 时间: 2016-5-23 23:21

不错啊! 一个字牛啊!

作者: haha3245 时间: 2016-9-14 12:54

哈哈,顶你了哦.

作者: 旅美学者 时间: 2016-9-18 17:53

小心大家盯上你哦

作者: 干细胞2014 时间: 2016-9-22 10:01

不错的东西持续关注

作者: highlight 时间: 2016-10-8 15:10

真是佩服得六体投地啊

作者: 昕昕 时间: 2016-10-22 16:14

呵呵,支持一下哈

作者: HongHong 时间: 2016-10-25 18:41

哈哈,这么多的人都回了,我敢不回吗?赶快回一个,很好的,我喜欢

作者: IPS干细胞 时间: 2016-11-9 14:54

干细胞研究还要面向临床

作者: 小小C 时间: 2016-11-9 20:27

彪悍的人生不需要解释。

作者: Whole 时间: 2016-11-14 16:18

(*^__^*) 嘻嘻……

作者: happyboy 时间: 2016-11-24 19:27

脂肪干细胞

作者: 未必温暖 时间: 2016-12-4 17:17

这贴子你会收藏吗

作者: ikiss 时间: 2016-12-4 21:05

角膜缘上皮干细胞

作者: 小丑的哭泣 时间: 2016-12-12 12:43

干细胞美容

作者: IPS干细胞 时间: 2017-1-17 13:10

昨晚多几分钟的准备，今天少几小时的麻烦。

作者: doors 时间: 2017-1-17 22:00

一个人最大的破产是绝望，最大的资产是希望。

作者: Diary 时间: 2017-2-7 21:18

干细胞从业人员

作者: marysyq 时间: 2017-3-2 08:18

顶你一下.

作者: ladybird 时间: 2017-3-14 08:01

…没我说话的余地…飘走

作者: 小丑的哭泣 时间: 2017-3-30 21:27

看贴回复是好习惯

作者: haha3245 时间: 2017-4-4 06:16

原来是这样

作者: bluesuns 时间: 2017-4-6 02:41

楼主good

作者: xiao2014 时间: 2017-4-13 10:43

表观遗传学

作者: 红旗 时间: 2017-4-23 20:59

好贴坏贴，一眼就看出去

作者: 某某人 时间: 2017-4-30 20:16

说的不错

作者: pcr 时间: 2017-5-5 11:01

活着，以死的姿态……

作者: dongmei 时间: 2017-5-15 13:10

青春就像卫生纸。看着挺多的，用着用着就不够了。

作者: 锦锦乐道 时间: 2017-7-2 13:27

心脏干细胞

作者: 修复者 时间: 2017-7-5 17:26

声明一下：本人看贴和回贴的规则，好贴必看，精华贴必回。

作者: 天蓝色 时间: 2017-7-7 22:56

真是天底下好事多多

作者: dd赤焰 时间: 2017-7-8 05:12

我等你哟!

作者: pspvp 时间: 2017-7-28 04:17

加油啊！偶一定会追随你左右，偶坚定此贴必然会起到抛砖引玉的作用~

作者: 水木清华 时间: 2017-8-4 06:43

顶下再看

作者: 化药所 时间: 2017-8-18 16:10

人气还要再提高

作者: pspvp 时间: 2017-8-25 10:50

自己知道了

作者: Whole 时间: 2017-9-21 07:06

我帮你喝喝

作者: IPS干细胞 时间: 2017-10-4 16:41

爷爷都是从孙子走过来的。

作者: 科研人 时间: 2017-10-17 11:27

努力,努力,再努力!!!!!!!!!!!

作者: 红旗 时间: 2017-12-1 13:17

人之所以能，是相信能。

作者: 剑啸寒 时间: 2017-12-3 16:34

看完了这么强的文章，我想说点什么，但是又不知道说什么好，想来想去只想

作者: 天蓝色 时间: 2017-12-18 11:10

感谢党和人民的关爱~~~

作者: 泡泡鱼 时间: 2017-12-20 20:55

不错啊! 一个字牛啊!

作者: dypnr 时间: 2017-12-31 12:10

顶也～

作者: 苹果天堂 时间: 2018-1-6 03:44

dc-cik nk

作者: dglove 时间: 2018-1-12 17:18

看完了这么强的文章，我想说点什么，但是又不知道说什么好，想来想去只想

作者: 蝶澈 时间: 2018-1-15 07:13

内皮祖细胞

作者: 碧湖冷月 时间: 2018-2-10 00:26

好困啊

作者: chongchong 时间: 2018-2-11 02:05

造血干细胞

作者: 老农爱科学 时间: 2018-3-23 18:20

每天早上起床都要看一遍“福布斯”富翁排行榜，如果上面没有我的名字，我就去上班……

作者: lalala 时间: 2018-3-26 04:26

琴棋书画不会，洗衣做饭嫌累。

作者: abc987 时间: 2018-3-31 05:01

我十目一行也还是看不懂啊

作者: 龙水生 时间: 2018-3-31 18:53

哈哈,这么多的人都回了,我敢不回吗?赶快回一个,很好的,我喜欢

作者: 生物小菜鸟 时间: 2018-4-17 17:57

努力~~各位。。。

作者: 生物小菜鸟 时间: 2018-4-22 17:27

我的啦嘿嘿

作者: 365wy 时间: 2018-4-26 05:51

楼主也是博士后吗

作者: MIYAGI 时间: 2018-5-19 08:27

就为赚分嘛

作者: 坛中酒 时间: 2018-5-19 16:09

小心大家盯上你哦

作者: 狂奔的蜗牛 时间: 2018-5-29 20:18

干细胞行业门户干细胞之家

作者: 求索迷茫 时间: 2018-6-12 09:01

必须顶

作者: 咕咚123 时间: 2018-7-12 19:28

对不起，我走错地方了，呵呵

作者: tuanzi 时间: 2018-7-20 16:30

支持一下吧

作者: txxxtyq 时间: 2018-8-17 22:57

好贴子好多啊

作者: 知足常乐 时间: 2018-8-31 05:40

一个人最大的破产是绝望，最大的资产是希望。

作者: myylove 时间: 2018-9-26 06:26

世界上那些最容易的事情中，拖延时间最不费力。

作者: 泡泡鱼 时间: 2018-10-6 10:59

继续查找干细胞研究资料

作者: 风云动 时间: 2018-10-7 15:27

dc-cik nk

作者: 分子工程师 时间: 2018-10-12 01:17

哦...............

作者: 某某人 时间: 2018-10-15 22:41

偶啥时才能熬出头啊.

作者: 某某人 时间: 2018-10-17 13:19

今天无聊来逛逛

作者: aliyun 时间: 2018-10-19 18:08

我在努力中

作者: Diary 时间: 2018-10-20 22:17

顶顶更健康，越顶吃的越香。

作者: 一个平凡人 时间: 2018-10-30 13:35

文笔流畅，修辞得体，深得魏晋诸朝遗风，更将唐风宋骨发扬得入木三分，能在有生之年看见楼主的这个帖子。实在是我三生之幸啊。

作者: 求索迷茫 时间: 2018-10-30 19:57

我起来了哈哈刚才迷了会

作者: heart10 时间: 2018-11-8 02:25

我在顶贴~！~

作者: 大小年 时间: 2018-12-23 01:09

我想要`~

作者: foxok 时间: 2019-1-12 20:43

干细胞分化技术

作者: laoli1999 时间: 2019-1-17 23:43

家财万贯还得回很多贴哦

作者: 3344555 时间: 2019-1-19 16:54

感觉好像在哪里看过了，汗~

作者: 快乐小郎 时间: 2019-1-25 06:21

加油啊！！！！顶哦!!!!!

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